Quantitative Determination of Cholesterol Hydroxylase Specificities by GC–MS/MS in Living Mammalian Cells

Cholesterol is oxygenated by a variety of cholesterol hydroxylases; oxysterols play diverse important roles in physiological and pathophysiological conditions by regulating several transcription factors and cell-surface receptors. Each oxysterol has distinct and overlapping functions. The expression of cholesterol hydroxylases is highly regulated, but their physiological and pathophysiological roles are not fully understood. Although the activity of cholesterol hydroxylases has been characterized biochemically using radiolabeled cholesterol as the substrate, their specificities remain to be comprehensively determined quantitatively. To better understand their roles, a highly sensitive method to measure the amount of various oxysterols synthesized by cholesterol hydroxylases in living mammalian cells is required. Our method described here, with gas chromatography coupled with tandem mass spectrometry (GC–MS/MS), can quantitatively determine a series of oxysterols endogenously synthesized by forced expression of one of the four major cholesterol hydroxylases—CH25H, CYP7A1, CYP27A1, and CYP46A1—or induction of CH25H expression by a physiological stimulus. This protocol can also simultaneously measure the amount of intermediate sterols, which serve as markers for cellular cholesterol synthesis activity. Key features • Allows measuring the amount of a variety of oxysterols synthesized endogenously by cholesterol hydroxylases using GC–MS/MS. • Comprehensive and quantitative analysis of cholesterol hydroxylase specificities in living mammalian cells. • Simultaneous quantification of intermediate sterols to assess cholesterol synthesis activity.

This protocol is used in: J. Biol.Chem.(2023), DOI: 10.1016/j.jbc.2022.102733Cholesterol is oxygenated by a variety of cholesterol hydroxylases; oxysterols play diverse important roles in physiological and pathophysiological conditions by regulating several transcription factors and cell-surface receptors.Each oxysterol has distinct and overlapping functions.The expression of cholesterol hydroxylases is highly regulated, but their physiological and pathophysiological roles are not fully understood.Although the activity of cholesterol hydroxylases has been characterized biochemically using radiolabeled cholesterol as the substrate, their specificities remain to be comprehensively determined quantitatively.To better understand their roles, a highly sensitive method to measure the amount of various oxysterols synthesized by cholesterol hydroxylases in living mammalian cells is required.Our method described here, with gas chromatography coupled with tandem mass spectrometry (GC-MS/MS), can quantitatively determine a series of oxysterols endogenously synthesized by forced expression of one of the four major cholesterol hydroxylases-CH25H, CYP7A1, CYP27A1, and CYP46A1-or induction of CH25H expression by a physiological stimulus.This protocol can also simultaneously measure the amount of intermediate sterols, which serve as markers for cellular cholesterol synthesis activity.

Graphical overview Background
Cholesterol plays diverse important biological roles, including function regulation of biological membranes and membrane proteins, and is the precursor for steroid hormones and bile acids; thus, cellular cholesterol homeostasis is tightly controlled by multiple mechanisms (Chang et al., 2006;Yamauchi and Rogers, 2018;Luo et al., 2020).Mammalian cells cannot break down the sterol backbone.Instead, cholesterol is converted to cholesteryl ester for storage (Chang et al., 2006) and to various oxysterols (Mutemberezi et al., 2016).Oxysterols exert different functions depending on the hydroxylation site(s).Multiple oxysterols whose side chain is hydroxylated, including 25-hydroxycholesterol (25-HC) and 27-HC, regulate cellular cholesterol homeostasis; they modulate two important transcription factors: sterol regulatory element-binding protein-2 (SREBP-2) and liver X receptor (LXR) (Gill et al., 2008; M.S. Brown et al., 2018).SREBP-2 transactivates most genes involved in cholesterol acquisition (biosynthesis and uptake) (Horton et al., 2002).On the other hand, LXR upregulates the expression of several ATPbinding cassette (ABC) transporters including ABCA1 and ABCG1 that mediate the export of excess cellular cholesterol (Tontonoz, 2011).Mechanistically, side-chain oxysterols bind to Insig-1 and Insig-2-retention factors for SREBP-2 in the endoplasmic reticulum (ER)-and protect them from proteasomal degradation, thereby inhibiting SREBP-2 activation.Side-chain oxysterols also serve as natural LXR ligands when added exogenously.

Published: Jan 20, 2024
A series of hydroxylases catalyze the site-specific hydroxylation of cholesterol (Mutemberezi et al., 2016).Although a variety of oxysterols have been identified in the body, 7α-HC, 27-HC, 24S-HC, and 25-HC are the most abundant.These four oxysterols are synthesized largely by CYP7A1, CYP27A1, CYP46A1, and CH25H, respectively, in the ER or mitochondria (Figure 1), while some of these hydroxylases also produce other oxysterols as minor products (Mutemberezi et al., 2016;Saito et al., 2023).Recent studies show that the expression of cholesterol hydroxylases is highly regulated in physiological and pathophysiological conditions (Cyster et al., 2014; A.J. Brown et al., 2021).CH25H expression and 25-HC biosynthesis are markedly upregulated upon infection in immune cells such as macrophages, and 25-HC itself exhibits anti-bacterial and anti-viral effects, protecting cells from infection (Bauman et al., 2009; S.Y. Liu et al., 2013;Zhou et al., 2020).25-HC can be further hydroxylated at the 7-position by the hydroxylase CYP7B1, generating 7α,25-dihydroxycholesterol (7α,25-diHC).This dihydroxycholesterol is a ligand for the G-protein coupled receptor GPR183 (also known as EBI2) involved in immune cell migration (C.Liu et al., 2011).Higher circulating 27-HC levels associate with the risk of estrogen receptor-positive breast cancer, since 27-HC serves as an endogenous selective estrogen receptor modulator (Nelson et al., 2013).CYP46A1 is a neuronspecific cholesterol hydroxylase that converts cholesterol into 24S-HC for eliminating excess cholesterol in the brain (Russell et al., 2009).CYP7A1 is a liver-specific hydroxylase that serves as the rate-limiting enzyme for bile acid synthesis (Russell, 2009).Although the activity of these cholesterol hydroxylases is biochemically studied using radiolabeled cholesterol as the substrate, comprehensive and quantitative characterization of their specificities remains poorly explored in living cells.Furthermore, precise measurement of the products of these cholesterol hydroxylases is crucial for better understanding their physiological and pathophysiological roles.Here, we provide a highly sensitive method using gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) to determine various oxysterols synthesized in living mammalian cells where a cholesterol hydroxylase is forcedly expressed or is upregulated by lipopolysaccharide, a component of Gram-negative bacteria known to upregulate CH25H expression in macrophages (Bauman et al., 2009).The current GC-MS/MS protocol enabled us to reveal cholesterol hydroxylase-specific production of oxysterols (Saito et al., 2023).Our protocol can also simultaneously determine the amount of intermediate sterols to monitor the activity of cholesterol biosynthesis.Accordingly, our GC-MS/MS-based sterol analysis, in combination with biochemical and gene expression studies, has suggested that side-chain oxysterols enzymatically synthesized within cells primarily regulate SREBP-2 but not LXR (Saito et al., 2023).

E. GC-MS/MS analysis
1. Inject 1 μL of sample or the standard mixture into a GCMS-TQ8040 NX equipped with a BPX5 GC column using an AOC-20i autosampler in splitless mode.Detailed conditions for the analysis are described in Table 1.Notes: a. Cholesterol-d7 and 25-hydroxycholesterol-d6 serve as internal standards for non-hydroxylated sterols and hydroxylated sterols, respectively.b.The quantification ions are used to identify and calculate the amount of each sterol.The confirmation ions are for assisting in the identification of respective sterols.Several sterols share the same precursor ions, quantification ions, and confirmation ions.When these sterols are not resolved, distinct precursor ions should be selected for the identification and quantification.c.Precursor ions for quantification and confirmation ions are carefully determined by the following criterion: higher m/z and stronger intensity.Collision energy is automatically calculated by the instrument.d.Retention times for each sterol become shorter after a column is cut for maintenance because it depends on the column length.

F. Cellular protein assay
1.After cellular lipid extraction (step B5), add 1 mL of 0.1 N NaOH (see Recipes) into each well and place at room temperature for 20-24 h to solubilize cellular protein.2. Mix solubilized proteins using a pipette and transfer 10 μL of a sample to a well of a 96-well plate.3. Add 150 μL of BCA assay reagent to each well, incubate the plate for 30 min at 37 °C, and record absorbance at 562 nm using a plate reader (SpectraMax M2e). 4. Determine the protein concentration of samples with bovine serum albumin as a standard.where E is extraction efficiency, C is the concentration (μg/mL) of cholesterol-d7 or 25-hydroxycholesterol-d6 detected by GC-MS/MS, V is the final volume (mL) of the sample (0.1 mL in this protocol), and I is the quantity (ng) of the internal standard added at the step B6 (100 ng of cholesterol-d7 or 10 ng of 25-hydroxycholeterol-d6, in this protocol).5. Calculate the original amounts of each sterol per sample using the appropriate extraction efficiency.
Note: The extraction efficiencies of cholesterol-d7 and 25-hydroxycholesterol-d6 are used to calculate the original amounts of non-hydroxysterols and oxysterols, respectively.6. Normalize the amounts of each sterol to cell protein per well (Figure 3).Although each method has advantages and limitations, the current GC-MS/MS protocol described above has several advantages over other methods.Since our method is highly sensitive, as 1 pg of sterols can be detected, lipids extracted from cells in a well of a 6-well plate (approximately 0.5-1 × 10 6 cells) are sufficient to measure a series of sterols, including oxysterols and intermediate sterols.The current GC-MS/MS analysis of cellular sterol is much more sensitive than our previous analysis with GC-MS, in which cellular lipids were extracted from a 100 mm dish or a whole 6-well plate (Yamauchi et al., 2007), while the amounts of cellular sterol contents detected are equivalent.Therefore, many samples can simultaneously be handled for studying multiple experimental conditions in a single assay.In addition, the run time for a sample in our method is approximately 15 min.Typical run times for sterol analysis by GC-MS and LC-MS/MS are 20-30 min and 12-20 min, respectively, depending on the columns and conditions used.Thus, our GC-MS/MS method is comparable to LC-MS/MS analysis concerning run time.Furthermore, GC-based methods generally show better chromatographic resolution than LC-MS/MS.The previous LC-MS/MS method was unable to resolve 7α-HC and 7β-HC (McDonald et al., 2012), whereas our GC-MS/MS protocol distinguished between these two oxysterols (Saito et al., 2023).As such, GC-MS/MS is the superior technology for analyzing small amounts of isomeric oxysterols, while a major limitation of LC-MS/MS methods is that such oxysterols tend to provide similar spectra.However, GC-MS/MS-based sterol analysis also has disadvantages.In contrast to LC-MS/MS methods, which do not often require derivatization (McDonald et al., 2012), sterol needs to be derivatized for GC-MS/MS analysis, which is a laborious and time-consuming process.In addition, since GC-MS/MS detects a compound of interest with MRM mode like LC-MS/MS, only target molecules with available standards can be measured.In summary, the current GC-MS/MS protocol provides a rapid and highly sensitive method for quantification of a series of oxysterols synthesized endogenously within cells, determining cholesterol hydroxylase activity quantitatively.

Figure 2 . 11 Published 1 .
Figure 2. Multiple reaction monitoring (MRM) chromatograms of sterols analyzed in this protocol.MRM chromatograms for each sterol are shown according to retention time.Quantification ions (black) and confirmation ions (pink and blue) are presented in each chromatogram.Black lines and pink and blue lines denote quantification ions and confirmation ions, respectively.

7 .
All data are represented by the mean ± S.D. of at least three independent biological replicates.Statistical analyses are performed with RStudio software by Student's t-test or one-way ANOVA with Dunnett or Tukey-Kramer post-hoc test.p < 0.05 is considered statistically significant.Advantages and limitations LC-MS/MS, GC-MS, and GC-MS/MS have been employed for sterol analysis, with GC-MS as the most traditional technique (Krone et al., 2010; McDonald et al., 2012; Griffiths et al., 2013; Saito et al., 2023).